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Lilium regale LrWRKY25 gene and application

A gene and lily technology, applied in the Minjiang lily LrWRKY25 gene and application field, can solve the problem that the biological function and transcriptional regulation mechanism are far from being elucidated.

Inactive Publication Date: 2019-03-19
YANGTZE NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that Lily Minjiang has strong resistance to lily fungal and viral diseases, and several stress-related genes have been obtained from Lily Minjiang, such as Lr14-3-3, LrPR10, LrbZIP1 and LrWRKY1 (Li H., et al., Sci. Hort. 2014, 168:9-16; He H., et al., Genes Genom. 2014, 36: 497-507; Zhang N.N., Genes Genom. et al., 2014 ,36:789-798; Han Q., et al., Sci.Hort.2016,198:370-378; Patent No. 201610001896.4), but there is no report on the regulation of plant growth and development by the WRKY transcription factor gene of Minjiang Lily , its biological function and transcriptional regulation mechanism are far from elucidated

Method used

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  • Lilium regale LrWRKY25 gene and application
  • Lilium regale LrWRKY25 gene and application
  • Lilium regale LrWRKY25 gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Lilium Minjiang LrWRKY25 full-length gene cloning and amino acid sequence analysis

[0036] (1) Cloning of the full-length sequence of the WRKY transcription factor gene LrWRKY25 in Lilium Minjiang

[0037] At present, the genome sequencing of Minjiang lily (Lilium regale) has not been completed. The research team obtained the reads sequence of LrWRKY25 through transcript sequencing analysis of different organizations in the early stage, and obtained its full-length sequence through sequence splicing; in order to verify the The LrWRKY25 full-length gene that actually exists in the gene, and then design specific primers (LrWRKY25-ATG-F and LrWRKY25-Stop-F) for PCR amplification analysis.

[0038] The leaf tissue of Minjiang Lily at the budding stage was taken as the material, and TRIzol was used to TM Plus RNA Purification Kit (12183555, Invitrogen TM ), total RNA was extracted according to the instructions, and DNase I (18047019, Invitrogen TM ) to remove ...

Embodiment 2

[0049] Example 2 Construction of recombinant expression vector P35S::GFP-LrWRKY25-NOS and analysis of subcellular localization

[0050] (1) Construction of P35S::GFP-LrWRKY25-NOS expression vector

[0051] According to the full-length gene sequence of Lilium Minjiang LrWRKY25 (SEQ ID NO.1), primers LrWRKY25-gfp-F and LrWRKY25-gfp-R were designed, and the seamless cloning (In-fusion) vector linker sequence and restriction site sequence were introduced into the primers . Using the TA-ligated positive clone plasmid in Example 1 as a template, LrWRKY25-gfp-F (forward primer) and LrWRKY25-gfp-R (reverse primer) were used as primers to perform specific amplification of the gene LrWRKY25.

[0052] The primer sequences are as follows:

[0053] LrWRKY25-gfp-F: 5'- TTCCCCGGGCTCGAG ATGACTTCATCCTCAGGAAGCATG-3';

[0054] LrWRKY25-gfp-R:5'- TTATCTAGATCCGGT GCAAGGCAAGGAGTCGAG-3';

[0055] Among them, the bold sequence of LrWRKY25-gfp-F is the Hind III restriction site, and the bo...

Embodiment 3

[0068] Example 3 Construction of recombinant expression vector P35S::LrWRKY25-NOS and genetic transformation of Arabidopsis

[0069] (1) Construction of P35S::LrWRKY25-NOS overexpression vector

[0070] According to the full-length gene sequence of Lilium Minjiang LrWRKY25 (SEQ ID NO.1), primers LrWRKY25-inf-F and LrWRKY25-inf-R were designed, and the seamless cloning (In-fusion) vector linker sequence and restriction site sequence were introduced into the primers . The specific amplification of the gene LrWRKY25 was performed using the TA-ligated positive clone plasmid in Example 1 as a template, and LrWRKY25-inf-F and LrWRKY25-inf-R as primers.

[0071] The primer sequences are as follows:

[0072] LrWRKY25-inf-F:5'- GGACTCTAGA ATGACTTCATCCTCAGGAAGCATG-3'

[0073] LrWRKY25-inf-R:5'- GATCGGGGAAATTC GCAAGGCAAGGAGTCGAG-3'

[0074] Among them, the bolded sequence of the forward primer LrWRKY25-inf-F is the BamHI restriction site, and the bolded sequence of the revers...

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Abstract

The invention discloses a lilium regale LrWRKY25 gene and application. The nucleotide sequence of the lilium regale LrWRKY25 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown asSEQ ID NO.2. An expression vector containing the LrWRKY25 gene is transferred into Arabidopsis plants, the obtained transgenic Arabidopsis plants show creep growth, and the original growth form of wild-type Arabidopsis is completely changed; and moreover, multiple rosette basal leaves grow from Arabidopsis stems in creep growth, and the leaf edge has a special form of regular projections. The lilium regale LrWRKY25 gene is very suitable for large-area cultivation and application in gardens and landscaping. Meanwhile, the lilium regale LrWRKY25 gene disclosed by the invention provides a theoretical basis and a technical support for preparing novel ground cover plants, and has potential application prospects.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a Minjiang lily LrWRKY25 gene and its application. Background technique [0002] Plant type is one of the important characters of ornamental plants, and breeding ideal plant type is an important goal of ornamental plant breeding. Prostrate growing plants are more suitable for the construction of ground cover landscape because of their low plant, small branch angle and close to the ground, and have broad application prospects in gardens. Research on creeping transgenic plants helps to expand its application value and further promote its application in landscaping and landscaping. [0003] WRKY is a family of transcription factors that are ubiquitous and conserved in plants. The earliest identified plant WRKY gene is the sweet potato (Impoea batatas) SPF1 (SPF1: SWEET POTATO FACTOR1) gene, whose gene product specifically recognizes the SP8 sequence i...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N5/10C07K14/415A01H5/12A01H6/20
CPCC07K14/415C12N15/8261
Inventor 符勇耀杨利平刘春渝徐文姬
Owner YANGTZE NORMAL UNIVERSITY
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